Loss of Slc12a2 specifically in pancreatic β-cells drives metabolic syndrome in mice

The risk of type-2 diabetes and cardiovascular disease is higher in subjects with metabolic syndrome, a cluster of clinical conditions characterized by obesity, impaired glucose metabolism, hyperinsulinemia, hyperlipidemia and hypertension. Diuretics are frequently used to treat hypertension in these patients, however, their use has long been associated with poor metabolic outcomes which cannot be fully explained by their diuretic effects. Here, we show that mice lacking the diuretic-sensitive Na+K+2Cl−cotransporter-1 Nkcc1 (Slc12a2) in insulin-secreting β-cells of the pancreatic islet (Nkcc1βKO) have reduced in vitro insulin responses to glucose. This is associated with islet hypoplasia at the expense of fewer and smaller β-cells. Remarkably, Nkcc1βKO mice excessively gain weight and progressive metabolic syndrome when fed a standard chow diet ad libitum. This is characterized by impaired hepatic insulin receptor activation and altered lipid metabolism. Indeed, overweight Nkcc1βKO but not lean mice had fasting and fed hyperglycemia, hypertriglyceridemia and non-alcoholic steatohepatitis. Notably, fasting hyperinsulinemia was detected earlier than hyperglycemia, insulin resistance, glucose intolerance and increased hepatic de novo gluconeogenesis. Therefore, our data provide evidence supporting the novel hypothesis that primary β-cell defects related to Nkcc1-regulated intracellular Cl−homeostasis and β-cell growth can result in the development of metabolic syndrome shedding light into additional potential mechanisms whereby chronic diuretic use may have adverse effects on metabolic homeostasis in susceptible individuals.


Introduction
Metabolic syndrome (MetS) is a common cluster of metabolic conditions reaching epidemic proportions. The main features include overweight/obesity, impaired glucose metabolism, hyperinsulinemia, hyperlipidemia and hypertension, which together strongly increase the risk for cardiovascular disease (CVD) and type-2 diabetes (T2D) [1][2][3]. In fact, the MetS is more frequent than T2D and its prevalence increases with age and overweight [4]. In addition, MetS together with obesity is considered the primary cause of non-alcoholic fatty liver disease (NAFLD) and its complications [5]. Therefore, preventing MetS constitutes a fundamental strategy to reduce CVD and T2D prevalence [1]. The etiology of the metabolic syndrome is complex. However, it is generally accepted that overeating calorie-dense diets rich in fats [6] and/or a sedentary life-style [7], is what eventually leads to increased adiposity, ectopic fat deposits, low-grade tissue inflammation, overweight/obesity and insulin resistance, the main drivers of the syndrome [8]. Current evidence suggests that insulin responses to feeding also play a role in the acute control of food intake [9] and that chronic hyperinsulinemia secondary to abnormal secretion/clearance is associated with a rise in fat mass accumulation [10]. It has been shown that lean individuals at risk of developing obesity have characteristically high and/or dynamically different insulin responses to nutrients, which persist or worsen during obesity [11,12]. Moreover, obese individuals also show abnormal pulsatile insulin secretion [13,14], all consistent with the notion that primary functional deficiencies in the islet secretory response to nutrients can contribute to the development of overweight and its complications including the MetS and T2D [15,16].
It is well recognized that pancreatic β-cells release insulin in a pulsatile manner [17] and in synchrony with intracellular Ca 2+ and/or metabolic oscillations [18,19]. Particularly, some of the mechanisms proposed to underlie β-cell Ca 2+ /metabolic oscillations and electrical bursting [20] appear unrelated to the canonical K ATP channel [21][22][23][24][25]. Indeed, a wide range of insulinotropic glucose concentrations promotes electrogenic Cl − fluxes while K ATP channel activity remains inhibited [26,27] whereas blocking these Cl − currents abolished membrane potential and Ca 2+ oscillations [28][29][30][31]. Chloride fluxes do require Cl − channels and some of them were independently implicated in β-cell function. For instance, volume-regulated anion channels (VRAC) [32-34], anoctamine-1 (Ano1) [30,31], the cystic fibrosis transmembrane conductance regulator (Cftr) [30,35] or the ionotropic receptors for γ-aminobutyric acid (GABA) [36,37] and glycine [38] all participate, to different extents, in β-cell excitability and insulin secretion. Independent of which Cl − channels are involved, secondary active Cl − loaders and extruders determine the non-equilibrium distribution of the anion and set the driving force for Cl − to flux through channels [39]. Inhibition of Cl − loaders such as Nkcc1 (Slc12a2) and others (Nkcc2, Slc12a1) with loop-diuretics bumetanide or furosemide impaired islet insulin secretion in vitro and resulted in glucose intolerance in different mouse models [40][41][42][43][44]. In addition, we have recently demonstrated that mice lacking a variant of the bumetanide-sensitive Nkcc2 (Nkcc2a, Slc12a1v1) exhibit abnormal insulin responses to glucose and develop hyperglycemia, glucose intolerance and insulin resistance [45]. In humans, diuretic treatment has been long associated with altered glucose homeostasis, insulin resistance [46-52] and worsening of the MetS [53]. Further, patients with functional deficiency of the thiazide-sensitive Cl − loader SLC12A3 are prone to overweight/obesity and the MetS [54][55][56][57][58]. At this point, it is important to keep in mind that the targets of diuretics, including Nkcc1, Nkcc2a and Slc12a3 are highly expressed in the kidney when compared to pancreatic β-cells [59,60] and that diuretics can inhibit islet insulin secretion directly [61][62][63]. Therefore, the diuretic-dependent worsening of metabolic homeostasis may, at least in part be mediated by extra-renal effects of these drugs.
Here, we generated a new mouse model constitutively lacking the Nkcc1 in β-cells (Nkcc1 βKO ) and present experimental evidence indicating spontaneous development of typical features of the metabolic syndrome in these mice. Indeed, by 30 weeks of age Nkcc1 βKO mice fed ad libitum a standard diet, are overweight, glucose intolerant, insulin resistant and develop non-alcoholic steatohepatitis (NASH). Therefore, our results provide a potential mechanistic explanation for the metabolic disturbances provoked by the chronic use of diuretics and a new pre-clinical mouse model to study the spontaneous development and progression of a syndrome considered a major risk factor of CVD and T2D.

Loss of Nkcc1 in β-cells reduces β-cell mass, insulin secretion and action
The results shown in Fig 2A demonstrate that islets from~22w old Nkcc1 βKO mice are less responsive to glucose than control islets (Ins1 Cre ). Importantly, bumetanide reduced the secretory response to glucose in control but not in Nkcc1 βKO islets, as expected for a highly specific inhibitor of Nkcc1 and Nkcc2. These data thus confirm functional elimination of the transporter in β-cells of Nkcc1 βKO islets. Note that the secretory response of islets from 8-10w old Nkcc1 βKO mice was reduced, albeit not significantly (S3A Fig). To determine if these findings relate to changes in islet β-cell number/size, a morphometric analysis was performed. The data demonstrates significantly reduced β-cell numbers (Fig 2B), volume ( Fig 2C) and mass ( Fig  2D) in 10w old Nkcc1 βKO relative to age-matched control mice (Nkcc1 lox/lox ). Accordingly, the islet-to-pancreas area ratio was significantly reduced in 10w old Nkcc1 βKO (Fig 2E) as well as the total number of β-cell clusters throughout the Nkcc1 βKO pancreas ( Fig 2F). As expected for normal mice, the β-cell morphometry parameters obtained in 10w old mice remained relatively unchanged in older mice. Since there were no significant differences found in α-cell count, volume, mass or area between mice of both genotypes (S3 Fig), together these data suggest that a combination of reduced β-cell volume and number contribute to the reduced Nkcc1 βKO islet secretory responses in vitro and overall reduction in pancreatic β-cell mass in Nkcc1 βKO mice.
Since normal insulin secretion activates hepatic insulin signaling to reduce de novo gluconeogenesis [65], we evaluated age-dependent hepatic insulin receptor (Insr)-mediated Akt phosphorylation and G6Pc expression in fed and 16h fasted 10-30w old Nkcc1 βKO mice. Fed control mice (Ins1 Cre ) showed the expected Insr-mediated increase in Akt phosphorylation (Fig 3A, left panel), which was barely detected in Nkcc1 βKO mice at all ages tested (Fig 3A, right  panel). Thus, in Nkcc1 βKO mice the response of the liver to food intake appears blunted. When mice were fasted, Akt phosphorylation was neither detected in control mice, as expected, nor in Nkcc1 βKO (Fig 3B). These data indicate reduced post-prandial hepatic Insr signaling in Nkcc1 βKO mice. However, expression levels of Insr were found reduced only in younger (10-20w) Nkcc1 βKO relative to controls and did not differ at 30 weeks of age in Nkcc1 βKO mice ( Fig 3C). Interestingly, G6Pc protein expression relative to β-actin remained unchanged in Nkcc1 βKO mice suggesting intact endogenous glucose production. However, as shown in Fig  3D, glucose responses to exogenous alanine increased in 30w old Nkcc1 βKO mice, thus suggesting age-dependent deterioration in the control of hepatic de novo gluconeogenesis.

Excess weight, increased fat mass and adipocyte hypertrophy in Nkcc1 βKO mice
Ad libitum chow-fed Nkcc1 βKO male mice significantly increased their body weight (BW) as they became older (Fig 4A), and this was not attributed to increased daily food intake (S4A  Notably, BW mass of Nkcc1 βKO did not significantly differ from that of control mice (Nkcc1 loxflox or Ins1 Cre ) from weaning (p19-21) up to~15w of age. Subsequently, Nkcc1 βKO were significantly heavier than control mice, without becoming overtly obese. As expected, weekly BW gain after weaning gradually declined with age in mice of both genotypes ( Fig 4B). However, the initial reduction in post-weaning BW gain of Nkcc1 βKO was followed by an episodic burst of accelerated BW gain, which preceded the onset of BW mass increase. Indeed, BW decline was significantly faster in Nkcc1 βKO mice during the first 6w of age. After that, BW gain increased significantly during the 9 th -11 th w of age and remained hastened thereafter, but this significant difference disappeared over time relative to control mice. Notably, an increasing proportion of Nkcc1 βKO mice began to lose weight between 25w and 30w of age ( Fig 4B) while their food intake also declined (S4A Fig). The results shown in Fig 4C and 4D confirm that Nkcc1 βKO BW accrual is due to a significant age-dependent increase in fat mass Expression pattern of insulin receptors (Insr, 95kDa), Akt (60kDa) and G6Pc (40kDa) and phospho-activation of Akt (pAkt) in liver extracts of 10w, 20w and 30w Nkcc1 βKO and control mice (Ins1 Cre ) fed (A) or fasted 16h (B). Shown are representative immunoblots loaded to represent 2 mice (n = 3-4 per genotype, age and condition). As loading control, we used β-actin (45kDa). C. Semi-quantitative densitometry analysis of hepatic Insr expression levels relative to β-actin expressed in arbitrary units (au). Shown are the mean ± SEM of 3 independent blots corresponding to 3 male mice of the indicated genotypes, ages and condition ( � p<0.05). D. Blood glucose excursions (mg/dl) during alanine tolerance tests (ATT) performed in 16h fasted Nkcc1 βKO and control mice (Ins1 Cre ) at the indicated ages (mean ± SEM, n = 9-10, � p<0.05). The areas under each curve (mg/ml/ min) are indicated as insets in D.
https://doi.org/10.1371/journal.pone.0279560.g003 Growth of Nkcc1 βKO and control (Nkcc1 lox/lox ) mice fed ad libitum a chow diet. Data recorded as net weekly BW mass (g) starting at weaning until mice reached 30w of age. Plotted are the mean ± SEM (n = 9-16, � p<0.01). B. Weekly BW gain (g/week) of Nkcc1 βKO and control (Nkcc1 lox/lox ) mice computed by subtracting BW at a given week age to that of the previous week. Each point represents data from a single mouse (n = 9-16, � p<0.01). C, D. Indicated are the mean ± SEM values corresponding to net fat mass (C, g) and lean mass (D, g) of Nkcc1 βKO and control (Nkcc1 lox/lox ) mice at the indicated ages (n = 9-16, � p<0.01). E. Mean cross sectional area (μm 2 ) of adipocytes morphometrically determined by analyzing retroperitoneal white fat tissue sections from 10w and 30w old Nkcc1 βKO and control (Nkcc1 lox/lox ) mice (n = 3). Each point represents the mean adipocyte area found in a single nonoverlapping digital image randomly taken from tissue sections (n = 6-9) of the indicated genotypes and ages ( � p<0.001). F. Relative mean adipocyte size distribution computed from the data in E. accumulation ( Fig 4C) rather than lean mass ( Fig 4D) or free/total body water content (S4B and S4C Fig). In a very consistent way, cross-sectional adipocyte areas and fat cell-size distribution in in white retroperitoneal fat tissue of 10w old Nkcc1 βKO mice were normal ( Fig 4E  and 4F, left panel). However, the mean adipocyte area and cell-size distribution were significantly expanded (Fig 4E), or shifted toward larger adipocytes, respectively, in 30w old Nkcc1 βKO mice ( Fig 4F, right panel). In fact, 90-95% of the adipocytes were smaller than~2000μm 2 in 10w and 30w old normal mice whereas~50% of all adipocytes in 30w old Nkcc1 βKO mice were larger than 2000μm 2 ( Fig 4F, left panel). Further, histological analysis of retroperitoneal white adipose tissue and pancreas of Nkcc1 βKO mice demonstrate infiltration of inflammatory cells (S4D and S4E Fig) and fat cell deposits (S4F and S4G Fig). Evidently, this increased local and ectopic fat mass accumulation and adipocyte hypertrophy account for the age-dependent increase in BW mass in Nkcc1 βKO mice.

Dyslipidemia and non-alcoholic fatty liver disease in Nkcc1 βKO mice
The results shown in Fig 5A demonstrate that plasma glycerol levels were significantly increased in 10w and 30w old Nkcc1 βKO , but hypertriglyceridemia only manifested later in 30w old Nkcc1 βKO mice ( Fig 5B) suggesting age-related deterioration of lipid metabolism. Importantly, Nkcc1 βKO did not develop larger livers than control mice discarding hepatomegaly ( Fig  5C). Within this context, total fat content was significantly elevated in the liver of 30w old Nkcc1 βKO relative to control ( Fig 5D,~9% and~4% w/w, respectively, � p<0.001) but not in 10w old Nkcc1 βKO mice (~3% w/w). Histological analysis revealed minimal and isolated micro vesicular steatosis in 10w old Nkcc1 βKO mice (Fig 5E and 5F) consistent with a normal score of 1 in the Kleiner's scale of NAFLD [66]. However, 30w old Nkcc1 βKO showed hepatocyte hypertrophy, micro/macro vesicular steatosis (

Age-dependent worsening of glycemic control in Nkcc1 βKO mice
Because 30w old Nkcc1 βKO mice developed NASH, we further tested glycemic control in these mice. Plasma insulin and blood glucose were determined in 10-30w old Nkcc1 βKO mice after their nocturnal feeding or after preventing it. Ten-week old Nkcc1 βKO showed minimal changes in fed or fasted plasma insulin and blood glucose levels relative to control mice (Ins1 Cre , Fig 6A and 6B, left panels). However, fasting plasma insulin levels increased in 20w old Nkcc1 βKO mice and both, fed/fasted plasma insulin and blood glucose were significantly higher in Nkcc1 βKO mice at 30w of age ( Fig 6A and 6B, center and right panels). Therefore, fasting hyperinsulinemia precedes the rise in blood glucose in Nkcc1 βKO mice whereas fed hyperinsulinemia and high blood glucose develop in older Nkcc1 βKO mice. Still, 30w old Nkcc1 βKO mice were not overtly hyperglycemic (e.g., >200 mg/dl) or hyperinsulinemic (e.g., >500 pmol/ L) indicating that the secretory dysfunction/β-cell loss in islets lacking Nkcc1 is insufficient to trigger T2D in chow-fed Nkcc1 βKO mice younger than~35w. Instead, it results in age-dependent worsening of glycemic control. In support of that conclusion, 10w and 20w old Nkcc1 βKO mice were normo-tolerant to exogenous glucose (Fig 6C, left and mid panel), whereas 30w Nkcc1 βKO mice were not (Fig 6C, right panel and Fig 6D). In addition, 30w old Nkcc1 βKO mice developed resistance to insulin-induced hypoglycemia (Fig 6E, right panel). Therefore, the excess weight goes in hand with increased fasting plasma insulin but appears before overt glucose intolerance and insulin resistance in Nkcc1 βKO mice.

Discussion
We present evidence indicating that Nkcc1 βKO mice develop a cluster of metabolic conditions compatible with the MetS. The metabolic features of Nkcc1 βKO mice are very similar to those found in the chow-fed Fatzo/Pco mouse model of MetS/NAFLD [67]. In fact, Fatzo/Pco mice became overweight/obese on a normal chow diet, independently of changes in energy intake [68] and had deficient insulin responses to oral glucose [67,69]. Although the mechanisms responsible for the phenotypes of the Fatzo/Pco mouse model are complex and likely polygenic, the ones present in Nkcc1 βKO mice appear directly related to the functional loss of Nkcc1 in β-cells. Indeed, i) Cre-mediated recombination of "floxed" alleles was only detected in βcells of Ins1 Cre :Nkcc1 lox/lox :Tomato (Fig 1A-1H) and Nkcc1 βKO mice (Fig 1I-1L), respectively; ii) Nkcc1 βKO islets exhibited expected recombination events and Nkcc1 transcript expression patterns (Fig 1M-1P); and iii) immunoreactive Cre was present only in β-cells of Ins1 Cre mice   [72,73] and brain in Ins1 Cre : Nkcc1 lox/lox :Tomato mouse model (S1E-S1I Fig) [74]. Along the same lines, Nkcc1 protein expression was intact in the choroid plexus (S1I Fig), a relevant finding because low levels of Ins1 gene activity were previously reported [75]. Moreover, since expression of Cre or "floxed" alleles did not alter Nkcc1 tissue expression patterns (S1 and S2 Figs), together these results support the conclusion that Nkcc1 βKO mice have lost Nkcc1 in β-cells, minimizing recent concerns related to the efficacy/efficiency of the Ins1 Cre line to eliminate target genes [76].
Consistent with the previous conclusion, the secretory function of 10w and 22w old Nkcc1 βKO islets is reduced by~25% and~50%, respectively (S2A and S3A Figs). Notably, disruption or chronic pharmacological inhibition of Nkcc1 does not eliminate insulin responses to glucose [35,44,45,62]. This is attributed to the fact that β-cells express a wide range of Cl − transporters and channels with potential overlapping and/or compensatory function, at least to some extent [39]. Nevertheless, our results suggest that β-cell Nkcc2a [45, 77] is minimally involved in the reduced secretory response of Nkcc1 βKO islets, because bumetanide did not reduce insulin secretion (S2A and S3A Figs). Along those lines, the participation of VRAC (Lrrc8a-e) [33,34,78] in the secretory phenotype of Nkcc1 βKO islets is expected to be limited because inhibition of Nkcc1 impairs β-cell volume regulation and VRAC activation [33,79]. Independent of the potential participation of the furosemide-sensitive Kcc2 (Slc12a5) [80] or that of other Cl − transporters or channels in the secretory response of Nkcc1 βKO islets, our data suggest that loss of Nkcc1 in β-cells results in a rather mild age-related secretory dysfunction. Further, the demonstration that 10w old Nkcc1 βKO islets were significantly smaller than control due to decreased β-cell number and volume (Fig 2B-2E) implies that the overall reduced in vitro secretory responses of these islets is also related, at least in part to their hypoplastic nature.
The mechanistic relationship between the loss of Nkcc1 in β-cells and reduced β-cell number/volume/size is intriguing but not surprising. It has been demonstrated that Nkcc1 participates in cell proliferation [81][82][83][84][85][86][87]. Actually, inhibition of Nkcc1 reduced the proliferative capacity of excitable neuronal progenitor cells by dampening the electrical activity of ionotropic GABA receptors [88], which are Cl − channels, whereas their activation increased mouse and human β-cell mass [89,90]. Our results demonstrating reduced number of β-cell clusters in the pancreas of 10-30w old Nkcc1 βKO mice ( Fig 2F) support a role for Nkcc1 as a potential regulator of progenitor cell proliferation, because these clusters are considered proto-islets [91]. In addition, our data demonstrating reduced cell volume in β-cells lacking Nkcc1 ( Fig  2C) are consistent with its role as a key regulator of mammalian cell volume [92] and, in particular, with biophysical [64,79], pharmacological [40, 93,94] and molecular [44] data directly implicating Nkcc1 in the regulation of β-cell volume/size. Therefore, the physiological metabolic consequences of losing Nkcc1 in β-cells are potentially related to reduced β-cell mass/volume/size due to dysregulated [Cl -] i , altered Cl − channel-mediated electrical activity or a combination of both. In fact, inhibition of β-cell Nkcc1 reduced glucose-induced β-cell electrical oscillations modulated by Cl − channels [29, 31] whereas isovolumetric circadian oscillations in [Cl -] i , determined by the activity of Nkcc1/ Kcc, established the frequency of action potential firings in electrically excitable cells [95]. Regardless of the underlying mechanisms, the age-related metabolic consequences of altered pulsatile/circadian insulin release are multiple [16]. These include hepatic Insr down-regulation, reduced insulin signaling and development of insulin resistance [96], impaired glucose tolerance, BW gain, dyslipidemia, liver fat accumulation and increased risk of NAFLD/NASH [15,97]. As we have shown, Nkcc1 βKO mice fed ad libitum a chow diet recapitulated most of the previous metabolic phenotypes in an age-dependent manner. At 10w of age, Nkcc1 βKO mice showed reduced hepatic Insr expression/signaling (Fig 3A and  3B), mild focal liver steatosis (Fig 5F) and reduced hepatic glycogen stores (S5G and S5H  Fig) considered early metabolic manifestations of deficient insulin-mediated responses in vivo [98,99]. Importantly, lean 10w old Nkcc1 βKO mice also showed increased plasma glycerol (Fig 5A), an early marker of lipolysis, altered triglyceride turnover [100,101] and a predictor of glucose intolerance/T2D in humans [102]. Consistently, 30w old Nkcc1 βKO mice developed glucose intolerance (Fig 6C and 6D), systemic insulin resistance (Fig 6E) and had increased responses to alanine (Fig 3D), a substrate almost exclusively used by the liver for de novo gluconeogenesis [45]. Further, older Nkcc1 βKO mice had fasting/fed hyperinsulinemia (Fig 6A), hyperglycemia ( Fig 6B) and developed overweight (Fig 4A-4C), severe dyslipidemia (Fig 5A and 5B) and NASH (Fig 5G and 5H and S5 Fig). Therefore, the agedependent metabolic phenotype of ad libitum chow-fed Nkcc1 βKO mice resembles most of the natural history of metabolic syndrome. In a physiological setting, our data rises the possibility that β-cell Nkcc1 may play a role in the natural decline of metabolic health associated with aging. In a clinical setting, our results may also provide a potential mechanism whereby chronic use of loop diuretics may worsen glucose homeostasis in patients with metabolic syndrome or susceptible to develop T2D.
In summary, our results demonstrate that the mild metabolic dysfunction of 10w old Nkcc1 βKO mice represents early phenotypic manifestations linked to a primary defect in β-cell function/proliferation/differentiation consequence of losing a diuretic-sensitive Cl − cotransporter. In addition, given that these phenotypes are not related to increased food intake, but precede the onset of overweight, it seems reasonable to conclude that the cascade of agerelated metabolic manifestations observed in these mice develop in parallel with BW gain, likely increasing the risk of developing T2D later in life.

Animals and housing
The Animal Care and Use Committee of Wright State University approved all methods involving mice, which were carried out in accordance to relevant guidelines and regulations. Mice were congenic on the C57BL/6J genetic background and crossed for~10 generations. Mice harboring loxP sites flanking exon 8-10 of the Slc12a2 gene (Nkcc1 lox/lox , provided by Dr. Christian A. Hübner, Jena University, Germany) were mated to Ins1 Cre mice [Jackson Labs stock 026801, B6(Cg)-Ins1 tm1.1(cre)Thor /J] constitutively expressing Cre recombinase only in pancreatic β-cells [103] to generate Ins1 Cre :Nkcc1 lox/lox mice (Nkcc1 βKO ). As control, we used the following homozygous mice: Ins1 Cre , Nkcc1 lox/lox , Nkcc1 WT (C57BL/6J) and the tdTomato reporter line [Jackson Labs stock 007909, B6.Cg-Gt(ROSA)26Sor tm9(CAG-tdTomato)Hze /J] to verify β-cell recombination of target alleles. In our hands, homozygous Cre expression in β-cells or the presence of "floxed" alleles in mice (Ins1 Cre and Nkcc1 lox/lox ) were not associated with changes in glucose homeostasis as determined by: basal/fed blood glucose, plasma insulin, and glucose and insulin tolerance, consistent with previous reports [103][104][105]. Mice had ad libitum access to water and a standard chow diet [Envigo, Teklad 22/5 Rodent Diet #8640 (3.0kCal/g, 54% carbohydrates, 29% proteins and 17% fats)], except when they were fasted. In that case, only water was provided. Housing conditions were set as 12:12h light (0630-1830h) and dark (1830-0630h) cycles with an ambient temperature of~22ºC. Data presented here correspond to experiments performed using male mice from~10 to~35 weeks (w) of age housed in groups as recently described [106].

Plasma biochemical studies, blood glucose and tolerance tests
Plasma was obtained after a 6h or 16h fasting period (0730-1330h or 1600-0800h, respectively) or at 0800h from mice fed ad libitum, by using heparinized glass capillaries (Scientific Glass, Rockwood, TN) and processed essentially as described [45]. Plasma triglycerides (TGs) and glycerol concentrations were determined by using commercially available kits (Cayman, Ann Harbor MI #10010303 and #10010755, respectively) and following the manufacturer's instructions. Plasma insulin was quantified by using an ultrasensitive ELISA (10-1247-01; Mercodia, Winston-Salem, NC). Whole blood glucose was determined with a glucometer (FreeStyle-Lite, Abbott, IL). Glucose and insulin tolerance tests (GTTs and ITTs, respectively) consisted in measuring 6h fasted glucose and serially 15, 30, 60 and 120 minutes after intraperitoneal administration of 2.0g/kg D-glucose or 0.75U/kg of human recombinant insulin (HumulinR Eli Lilly, Indianapolis, IN). Alanine tolerance tests (ATTs) were performed in 16h fasted mice as described [45].

Primary islets and insulin secretion
Mice were deeply and terminally anesthetized (Euthasol 1 , ip 150mg/kg) and pancreas tissues processed to isolate islets by using the collagenase method as previously described [45]. Islets were handpicked into individual wells of 12-well plates with mesh inserts [15 islet equivalents (iEq)/well] containing KRBH (in mM: 118.5 NaCl, 2.5 CaCl 2 , 1.2 KH 2 PO 4 , 4.7 KCl, 25 NaHCO 3 , 1.2 MgSO 4 , 10 HEPES and 0.1% BSA pH 7.4) plus 3.3mM glucose. The mesh inserts containing islets were transferred to new wells containing KRBH+3.3mM glucose and incubated at 37ºC (5% CO 2 ) for 30 minutes, a step repeated once more. The islets were then transferred into their respective experimental wells containing KRBH+5.5mM or +12.5mM glucose plus vehicle (DMSO) or bumetanide (#B3023, Sigma Chem Co. Saint Louis, MO) for 1h at 37ºC (5% CO 2 ). Islets were transferred into new wells containing KRBH+12.5mM glucose plus vehicle or drugs, incubated 1h at 37ºC (5% CO 2 ) and transferred to new wells containing acidified ethanol. The KRBH from experimental wells was frozen at -20ºC for further analysis. Insulin content or secreted into the media was estimated using ELISA (10-1247-01, Mercodia, Salem, NC). Results are expressed as the ratio between secreted insulin and the sum of secreted and islet insulin content.

Body composition and liver fat content analysis
Total body fat, lean mass and body water were determined in live mice by using the whole body quantitative magnetic resonance imaging (QMRI) analyzer EchoMRI-500™ system (EchoMRI LLC, Echo Medical Systems, Houston TX) as described [109]. Mice were then sacrificed by decapitation to determine hepatic fat content (w/w) by using the gravimetric method of Bligh and Dyer [110]. Briefly, liver samples were homogenized in chloroform:methanol: water (2:2:1.8) using a manual glass/glass homogenizer on ice. The homogenate was centrifuged at 625×g and the organic phase collected and washed once with double distilled water to help with phase separation. The chloroform phase containing extracted fat was vacuum-dried in a rotary evaporator (SC110A SpeedVac Plus) at high drying rate. The residue was then analytically weighed (Mettler Toledo, AE100).

Western blotting
Tissues were weighed and immediately submerged in liquid nitrogen or immediately processed to extract proteins. Briefly, liver tissues were minced and quickly homogenized at 4ºC in a glass/glass homogenizer (Wheaton 15ml) containing Radioimmunoprecipitation assay (RIPA) lysis buffer (Sigma, Saint Louis, MO, #R3792) supplemented with phenylmethylsulfonyl fluoride (PMSF) and a protease/phosphatase inhibitor cocktail (Thermo Sci., Waltham, MA, #78443) to a proportion of 3ml RIPA per gm of tissue. Tissue lysates were transferred onto a 15ml conical tube (Fisher Scientific, Corning #430790) and re-homogenized by passing the lysate~20 times through 18-gauge needles attached to a 5ml plastic syringe followed bỹ 20 more strokes through 21-gauge needles. Protein concentration in tissue extracts was determined by using the Coomassie-Bradford protein assay kit (Thermo Sci., Waltham, MA, #23200) following the instructions of the manufacturer. Up to 50μg of total proteins boiled 5min in denaturing loading buffer (Novex #2107345) were resolved in duplicates by polyacrylamide gel electrophoresis (PAGE) by using Bolt™ 4-12%, Bis-Tris pre-casted gels (Thermo-Fisher Sci., #NW04120). Molecular weights were estimated by using pre-stained protein standards (SeeBlue Plus 2, ThermoFisher Sci., #LC5925). Gels were run at 130V for 35min in 2-(N-morpholino)-ethanesulfonic (MES) acid buffer (ThermoFisher Sci., #B000202), removed and soaked in 20% ethanol for 5 mins before transferring them onto pre-assembled transfer PVDF stacks (iBolt Transfer Stack, ThermoFisher Sci.). Proteins were electroblotted onto PDVF membranes by using a dry blotting system (Life Technologies, iBolt 2) and then incubated in blocking buffer (SuperBlock T20, ThermoFisher Sci. #37516) overnight at 4ºC. Membranes were washed three times for 10min in Tris-buffered saline (TBS) plus Tween 20 (TBST) and exposed to primary antibodies for 48h at 4ºC with gentle rocking. Membranes were then washed four times for 10min in TBST and exposed to relevant secondary antibodies for 1h at room temperature. After washing excess antibodies, antigen/antibody reactions were developed by chemiluminescence (Pierce West Pico Plus, ThermoFisher Sci., #34577). Images were taken using ChemiDoc Imaging System (Bio-Rad, Hercules, CA). Membranes were either stripped off the first antibody and reblotted, or new blots were produced when different antibodies were needed to detect proteins of similar molecular weight. The primary antibodies used were directed against: insulin receptor β-subunit (Insr), the S/T protein kinase Akt and its active version pAkt phosphorylated in S 473 (rabbit, Cell Signaling #3025, #9272 and #9271, respectively), the catalytic subunit of glucose-6-phosphatase G6Pc (rabbit, Abcam ab83690) and β-actin (mouse, Developmental Studies Hybridoma Bank #528068). Secondary HRP-conjugated antibodies used were: anti-rabbit IgG and anti-mouse IgM (Jackson Immunoresearch, PA, #711-035-152 and #315-035-049, respectively).

Energy intake
Net 24h food intake was recorded in individually identifiable group-housed mice at 10w, 20w and 30w of age. Data was collected during 2 consecutive weeks after a week of acclimation in a metabolic cage equipped to record the feeding behavior of mice in real-time (Feed and Water intake activity monitor system HM-2, MBRose, Faaborg, Denmark). The overall settings, calibration and design of these experiments have been described in detail elsewhere [106]. The feeding microstructure/dynamics and ambulatory activity of Nkcc1 βKO and Ins1 Cre shall be reported in a forthcoming manuscript.

Statistics
Results are represented as mean values ± SEM, with the number of individual points (n) indicated. Statistical significance for a p value <0.05 between groups was obtained by applying one-way or two-way analyses of variance (ANOVA), as appropriate, followed by the Tukey-Kramer post-hoc test. Statistical analyses were conducted by using GraphPad Prism v7 (Graph-Pad Software Inc., San Diego, CA, USA). Normal distribution and homogeneity of data variance were tested using Shapiro-Wilk and F-tests, respectively. (TIF) S1 Raw images. Original full-size PCR and RT-PCR gels. The red rectangles on top of gels A and B represent the cropped areas used to build Fig 1N and 1P (B), respectively, in the main text. A. Original gel of genomic PCR experiments using DNA (+DNA) or not (-DNA) as templates. Shown are amplified DNA fragments of expected sizes obtained by using the primer sets indicated in Fig 1M. Also shown are additional control reactions performed by using primers designed to amplify 123bp of genomic sequences corresponding to the Slc12a5 gene. B. Original full-size RT-PCR gel showing bands of expected sizes corresponding to Cre (390bp) and Nkcc1 transcripts (400bp) amplified from total RNA purified from Nkcc1 βKO (lanes a and b)